PROJECTSUMMARY/ABSTRACT Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and mediates diverse functions including sensory and motor processing, as well as learning and memory. The energeticdemandofthisexcitatoryactivityismetbyalocalizedincreaseinbloodflow.Althoughthisincrease inbloodflowisimportanttosupporttheenergydemandsofneuraltissueandrepresentsthebasisofthesignal monitoredwithfunctionalmagneticresonanceimaging(fMRI),themechanism(s)thatunderliethiseffectremain unresolved. Unlike other classical neurotransmitters, that are directly recycled into the presynaptic nerve terminal, most glutamate is cleared into astrocytes. This clearance is mediated by two Na+-dependent transporters, called GLT-1 and GLAST (or EAAT2 and EAAT1, respectively). These transporters are almost exclusivelyexpressedbyastrocytesandenrichedonfineastrocyteprocessesnearsynapsesandonastrocyte endfeet. We have recently shown that glutamate transporters, Na+/Ca2+ exchangers, and mitochondria are functionally coupled to one another in astrocyte processes. We provide a strong scientific premise for the hypothesisthatincreasesinbloodflowuponneuronalactivationareduetoglutamatetransportintoastrocytes. InstudiesproposedinSpecificAim1,wewilluse2-photonimagingcombinedwithpharmacologicandgenetic manipulations to test the hypothesis that glutamate transport and Na+/Ca2+ exchange increase calcium in astrocyte endfeet and that this increase in calcium is necessary for stimulus-evoked increases in arteriole diameterinvivo.Normally,excitatoryactivitycausesanincreaseinbloodflow,butundersomecircumstances, theresponsebecomesinverted.InSpecificAim2,wewilltestthehypothesisthatpreventingmitochondriafrom dockinginastrocyteprocesses/endfeetresultsinexaggeratedstimulus-evokedcalciumsignalinginendfeetand inversionoftheneurovascularresponse.Afterastroke,decreasesinbloodflowextendbeyondtheoccluded vessel. In Specific Aim 3, we will test the hypothesis that focal ischemia results ina lossof mitochondria from astrocyte processes, exaggerated stimulus-evoked calcium signaling in endfeet, and inversion of the neurovascular response in the penumbra These studies will define a novel mechanism by which neuronal activitycausesanincreaseinneuronalbloodflow,willdefineanovelmechanismbywhichthisresponseinverts, andadeterminehowthesephenomenacontributetodysregulatedbloodflowobservedafterstroke.